Starch is a polymer of glucose linked by α(1–4) linkages to give linear chains, H which are joined by α(1–6) linkages resulting in branches in the polymer. Normal starch consists of about 75% amylopectin, a highly-branched molecule, and 25% of amylose, a primarily linear molecule. These polymers are organized into an insoluble granule within cereal seeds. Starch granules are highly organized, containing of a series of concentric spheres consisting of alternating crystalline and amorphous regions (Cameron and Donald, 1992).
Starch is synthesized by a series of enzymatic reactions (for review, see Martin and Smith, 1995; Myers et al., 2000). Glucose-1-phosphate is first activated to ADP-glucose by the enzyme ADP-glucose pyrophosphorylase (ADPGPP). This enzyme is heavily regulated and is thought to control the flux of carbon into starch biosynthesis, and therefore the amount of starch made. The structure of starch is determined by the subsequent enzymes in the pathway. Starch synthases (SS) catalyze the polymerization of ADP-glucose to produce a linear glucan polymer. Branches are introduced into this polymer by starch branching enzymes (SBE). Starch debranching enzymes (SDBE) contribute to starch structure by removing excess branches, which may help to establish the pattern of crystalline and amorphous regions within the granule.
Starch structures differ in different species. For example, barley and wheat amylopectins have larger portions of short branch chains (6 to 14 glucose units), have proportionally fewer branch chains of 11 to 22 glucose units and >40 glucose units, and larger proportions of branch linkages located within the crystalline region than maize amylopectin (Jane et al., 1999; Song and Jane, 2000). It is the starch structure that determines the functionality of starch.
The higher starch yield of corn as a C-4 crop makes cornstarch the most economic commodity in the world. Starch is easily isolated from corn seeds during milling process as compared to barley or wheat whose awns or gluten makes starch separation more difficult. In addition, the higher phospholipid content of barley and wheat starches restricts starch swelling and paste viscosity. However, barley or wheat starches have lower gelatinization temperatures than cornstarch, and thus require less energy for processing and cooking (Jane et al., 1999). Compared to cornstarch, barley or wheat starch has a lower retrogradation rate during storage, which translates into better paste stability and prolonged shelf life (Shi and Seib, 1992,1995; Yuan et al., 1993; Jane et al., 1999). Barley and wheat starches are also easier to digest by enzyme and animals than cornstarch, which can result in faster glucose production from starch and increased energy availability to livestock. This is particularly beneficial to young and small animals such as baby chicks that have shorter digestive tracts.
Chemical modifications of starch (e.g. chemical derivatives) are commonly used in the wet-milling industry to reduce the gelatinization temperature and retrogradation rate. Chemical modification processes are energy demanding, requiring large quantities of chemical reagents and salts during the reaction and washing and drying after the reaction. Recovery of byproducts, unreacted reagents, and salts (e.g. sodium sulfate) from wastewater is costly and has the potential to cause environmental pollution.
Genetic modification of starch structure provides an attractive alternative to improve functionality. It was estimated that genetic modification of cornstarch structure and functionality could add value $1.25 billion per year, with an average added value of $5.80 per bushel (Johnson et al., 1999a). An increase of cornstarch digestibility by 10% for livestock feed would add another $1.44 billion per year, with an average added value of $0.21 per bushel (Johnson et al., 1999b). In addition, genetically modified corn may be used to make possible new starch products (such as biodegradable plastics) and create new markets.
Genes or cDNAs of most starch biosynthetic enzymes have been cloned in corn, potato, barley, and wheat. They can be used to over- or under-express these enzymes by using sense or antisense transgenes. Expression of an E. coli ADPGPP in potato tubers increased starch content by 35% (Stark et al., 1992). Transformation of the amylose deficient amf mutant of potato with the granule-bound starch synthase (GBSS) gene led to amylose synthesis (Flipse et al., 1994), whereas various levels of reduction in GBSS protein and amylose were observed in transgenic rice endosperm with the antisense GBSS gene connected to the rice GBSS promoter or maize Adhl promoter (Terada et al., 2000). Antisense inhibition of two soluble SS of potato individually or simultaneously led to distorted starch granules and an enrichment in short chains and a reduction in longer chains of amylopectin (Edwards et al., 1999; Lloyd et al., 1999). Expression of an E. coli branching enzyme in tubers of amylose-free potato showed an increased branching degree and more short chains (16 glucose-residues or less) of the amylopectin (Kortstee et al., 1996). The antisense inhibition of the main SBE in potato tubers (SBE B) resulted in novel starch characteristics but not in an increased amylose level (Safford et al., 1998). However, transgenic potato plants expressing an antisense SBE A (the minor form of SBE) RNA increased the average chain length of amylopectin, resulting an moderate increase in apparent amylose content up to 38% (Jobling et al., 1999). Antisense inhibition of both SBE A and B simultaneously led to the production of potato starch with high-amylose and no amylopectin (Schwall et al., 2000).
There is a need in the art for a method of producing cornstarch combining the advantages and avoiding the disadvantages of corn and barley starches using genetic engineering.